U.S. patent number 7,368,550 [Application Number 11/388,339] was granted by the patent office on 2008-05-06 for phosphorus protecting groups.
This patent grant is currently assigned to Agilent Technologies, Inc.. Invention is credited to Marvin H Caruthers, Douglas J Dellinger, Geraldine Dellinger, Zoltan Timar.
United States Patent |
7,368,550 |
Dellinger , et al. |
May 6, 2008 |
**Please see images for:
( Certificate of Correction ) ** |
Phosphorus protecting groups
Abstract
Compounds having a phosphorus group structure of: ##STR00001##
wherein: R is alkyl, modified lower alkyl; and R.sup.i and R.sup.i
are each independently H, alkyl, modified alkyl, or aryl; are
provided. Also provided are polynucleotide compositions that
include these compounds and methods of using the compounds in
synthesis of the same.
Inventors: |
Dellinger; Douglas J (Boulder,
CO), Timar; Zoltan (Boulder, CO), Dellinger;
Geraldine (Boulder, CO), Caruthers; Marvin H (Boulder,
CO) |
Assignee: |
Agilent Technologies, Inc.
(Santa Clara, CA)
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Family
ID: |
37997241 |
Appl.
No.: |
11/388,339 |
Filed: |
March 23, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20070099859 A1 |
May 3, 2007 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60731723 |
Oct 31, 2005 |
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Current U.S.
Class: |
536/23.1;
536/25.31; 536/26.7; 536/26.8 |
Current CPC
Class: |
C07H
21/00 (20130101); Y02P 20/55 (20151101) |
Current International
Class: |
C07H
21/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Crane; L. E.
Parent Case Text
RELATED APPLICATIONS
Related subject matter is disclosed in U.S. Patent Application
filed by Dellinger et al. entitled "Monomer Compositions for the
Synthesis of Polynucleotides, Methods of Synthesis, and Methods of
Deprotection" and, now U.S. patent application Ser. No. 11/387,388;
U.S. Patent Application filed by Dellinger et al. entitled "Monomer
Compositions for the Synthesis of Polynucleotides, Methods of
Synthesis, and Methods of Deprotection" and, now U.S. patent
application Ser. No. 11/388,112; U.S. Patent Application filed by
Dellinger et al. entitled "Solutions, Methods, and Processes for
Deprotection of Polynucleotides" and, now U.S. patent application
Ser. No. 11/387,369; U.S. Patent Application filed by Dellinger et
al. entitled "Use of Mildly Basic Solutions of Peroxyanions for the
Post-Synthesis Deprotection of RNA Molecules and Novel Monomer
Compositions for the Synthesis of RNA" and, now U.S. Provisional
Application No. 60/785,130; U.S. Patent Application filed by
Dellinger et al. entitled "Cleavable Linkers for Polynucleotides"
and, now U.S. patent application Ser. No. 11/389,388; U.S. Patent
Application filed by Dellinger et al. entitled "Thiocarbonate
Linkers for Polynucleotides" and, now U.S. patent application Ser.
No. 11/751,692; all above-mentioned patent applications filed on
the same day as the present application. Related subject matter is
also disclosed in U.S. Provisional Patent Application filed on Oct.
31, 2005 by Dellinger et al. entitled "Methods for Deprotecting
Polynucleotides" having Ser. No. 60/731,723 filed Oct. 31, 2005
Claims
What is claimed is:
1. A compound having a structure selected from structure (I) or
structure (II): ##STR00028## or a salt thereof, wherein:
HeteroBase* is an optionally protected nucleobase; A is H, OH, or a
protected 2'-hydroxyl group; R is a modified lower alkyl, or alkyl;
R.sup.i and R.sup.ii are each independently H, alkyl, modified
alkyl, or aryl; R.sup.iii and R.sup.iv are each independently lower
alkyl, or R.sup.iii and R.sup.iv taken together are cycloalkyl; and
R.sup.v is H, a hydroxyl protecting group, a nucleotide moiety, or
an oligonucleotide moiety.
2. The compound of claim 1, wherein A is a protected 2'-hydroxyl
group, R is methyl, ethyl, n-propyl, or isopropyl; R.sup.i and
R.sup.ii are each independently H, methyl, ethyl, n-propyl, or
isopropyl; R.sup.iii and R.sup.iv are each isopropyl; and R.sup.v
is a hydroxyl protecting group.
3. The compound of claim 1, wherein R.sup.v is a hydroxyl
protecting group.
4. A polynucleotide, the polynucleotide comprising at least one
nucleotide subunit having the structure (III): ##STR00029##
wherein: HeteroBase* is an optionally protected nucleobase; A is H,
OH, or a protected 2'-hydroxyl group; R is a modified lower alkyl,
or alkyl; R.sup.i and R.sup.ii are each independently H, alkyl,
modified alkyl, or aryl; and the broken lines indicate sites of
attachment to the remainder of the polynucleotide.
5. The polynucleotide of claim 4, wherein A is a protected
2'-hydroxyl group, R is selected from methyl, ethyl, n-propyl, or
isopropyl; and R.sup.i and R.sup.ii are each independently H,
methyl, ethyl, n-propyl, or isopropyl.
6. A polynucleotide, the polynucleotide comprising a plurality of
nucleotide subunits, at least one of said plurality of nucleotide
subunits bound to a phosphorus protecting group, the phosphorus
protecting group having the structure (V): ##STR00030## wherein: R
is modified lower alkyl, or alkyl; R.sup.i and R.sup.ii are each
independently H or lower alkyl; and the broken line indicates a
bond to said at least one of said plurality of nucleotide
subunits.
7. The polynucleotide of claim 6, wherein R is a methyl, ethyl,
n-propyl, or isopropyl; and R1 and R'' are each independently H,
methyl, ethyl, n-propyl, or isopropyl.
8. The polynucleotide of claim 6, wherein each one of at least 50%
of the plurality of nucleotide subunits is bound to a respective
phosphorus protecting group, each respective phosphorus protecting
group having the structure (V).
9. The polynucleotide of claim 6, wherein the polynucleotide is DNA
or RNA.
10. A method of deprotecting a protected polynucleotide, the method
comprising; contacting the polynucleotide with a solution
comprising an .alpha.-effect nucleophile, wherein the
polynucleotide comprises a plurality of nucleotide subunits, at
least one of said plurality of nucleotide subunits bound to a
phosphorus protecting group, the phosphorus protecting group having
the structure (V): ##STR00031## wherein: R is a modified lower
alkyl, or alkyl; R.sup.i and R.sup.ii are each independently H,
alkyl, modified alkyl, or aryl; and the broken line indicates a
bond to said at least one of said plurality of nucleotide subunits;
said contacting resulting in cleavage of the phosphorus protecting
group from said at least one of said plurality of nucleotide
subunits.
11. The method of claim 10 wherein the solution is at a pH of about
6 to about 11.
12. The method of claim 10 wherein the .alpha.-effect nucleophile
has a pK.sub.a in the range of about 4 to 13.
13. The method of claim 10 wherein the solution comprising the
.alpha.-effect nucleophile is a solution comprising one or more
species selected from hydrogen peroxide, a peracid, a perboric
acid, an alkylperoxide, a hydroperoxide, a butylhyd roperoxide, a
benzylhyd roperoxide, a phenylhydroperoxide, a cumene
hydroperoxide, performic acid, peracetic acid, perbenzoic acid, a
substituted perbenzoic acid, chloroperbenzoic acid, perbutyric
acid, tertiary-butylperoxybenzoic acid, decanediperoxoic acid and
corresponding salts of said species.
14. The method of claim 10 wherein the solution comprising the
.alpha.-effect nucleophile is a solution comprising one or more
species selected from hydrogen peroxide, salts of hydrogen
peroxide, and mixtures of hydrogen peroxide and performic acid.
15. The method of claim 10 wherein the .alpha.-effect nucleophile
is formed in situ by a reaction of hydrogen peroxide and a
carboxylic acid or carboxylic acid salt.
16. The method of claim 10 wherein the .alpha.-effect nucleophile
has a pK.sub.a of about 4 to 13 and the solution is at a pH in the
range from about 6 to about 11.
17. The method of claim 10 wherein the cleavage of the phosphorus
protecting group results in a deprotected polynucleotide.
18. The method of claim 10 wherein the cleavage of the phosphorus
protecting group results in a solution of deprotected
polynucleotide, the method further comprising adding an alcohol to
the solution of deprotected polynucleotide to result in
precipitation of the deprotected polynucleotide, and recovering the
precipitated polynucleotide.
19. The method of claim 10 further comprising wherein the protected
polynucleotide is attached to a substrate via a cleavable linker.
Description
FIELD OF THE INVENTION
The invention relates generally to nucleic acid chemistry. More
particularly, the invention relates to providing protecting groups
useful in polynucleotide synthesis, as well as other uses.
BACKGROUND OF THE INVENTION
Over the past twenty years, the method of choice for the chemical
synthesis of oligonucleotides (ONs) has been the phosphoramidite
four-step process which utilizes the reaction of deoxynucleoside
phosphoramidites with a solid phase tethered nucleoside or
oligonucleotide (Letsinger, R. L.; Lunsford, W. B. J. Am. Chem.
Soc. 1976, 98, 3655-3661; Beaucage, S. L.; Caruthers, M. H.
Tetrahedron Lett. 1981, 22, 1859-1862; Matteucci, M. D.; Caruthers,
M. H. J. Am. Chem. Soc. 1981, 103, 3186-3191).
##STR00002## Initially the 5'-O-dimethoxytrityl (DMT) group is
removed from a deoxynucleoside linked to the polymer support. Step
2, elongation of a growing oligodeoxynucleotide, occurs via the
initial formation of a phosphite triester intemucleotide bond. This
reaction product is first treated with a capping agent designed to
esterify failure sequences and cleave phosphite reaction products
on the heterocyclic bases. The nascent phosphite intemucleotide
linkage is then oxidized to the corresponding phosphotriester. In
the final step of each cycle, the DMT group is removed from the
growing oligonucleotide using a large excess of a weak acid,
trichloroacetic acid (TCA), in an organic solvent. Further
repetitions of this four-step process generate the ON of desired
length and sequence. The final product is cleaved from the solid
phase and obtained free of base and the .beta.-cyanoethylphosphate
(Sinha, N. D.; Biernat, J.; Koster, H., Tetrahedron Lett. 1983, 24,
5843-5846) protecting groups by treatment of the support with
concentrated ammonium hydroxide, methyl amine or other
nucleophillic strong bases (Ogilvie, K. K.; Theriault, N. Y.;
Seifert, J-M.; Pon, R. T.; Nemer, J. J. Can. J Chem. 1980, 58,
2686-26930).
We have recently developed a new method of deprotection of
oligonucleotides that does not require strong bases like ammonia or
methyl amine. This method utilizes the strong nucleophilicity of
peroxyanions at mildly basic pH. This is especially applicable to
the chemical synthesis of oligoribonucleotides (RNA). Since this
method has many significant advantages for the deprotection of
oligonucleotides, it is especially appropriate to develop
protecting groups that are specifically designed to utilize these
novel deprotection conditions. Although many of the standard
protecting groups in the prior art can be removed using these novel
conditions, those standard protecting groups were optimized for
removal using strong bases. In addition, several of the standard
protecting groups require strong bases. A clear example of this is
the .beta.-cyanoethylphosphate protecting group. This group is
typically removed by a .beta.-elimination reaction (see U.S. Pat.
Re34,069 to Koster et al.). The typical .beta.-elimination reaction
occurs by having an electron withdrawing group in the
.alpha.-position to a methylene carbon. This makes the protons of
the .alpha.-methylene group acidic and they can thereby be removed
using a strong base. The compound then eliminates the phosphate in
the .beta.-position and forms an alkene such as acryonitrile.
##STR00003##
Although this works well for the use of a strong base like ammonia,
we typically use peroxyanion solutions at pH conditions below 11.
At this pH the proton cannot easily be abstracted, and these
.beta.-elminination reactions are not well suited for use with
peroxyanions.
The use of strongly electron withdrawing groups such as the
cyanoethyl groups has the additional disadvantage of deactivating
the phosphoramidite reagent toward coupling of the intemucleotide
bond. This is especially important in the chemical synthesis of
RNA. In typical RNA synthesis the 2'-hydroxyl is protected creating
additional inhibition of coupling by crowding around the reactive
phosphorus center.
##STR00004## As shown above, protonation by azole acid catalysts
followed by nitrogen exchange converts the phosphite species to
several highly active electrophiles. An electron withdrawing
protecting group can significantly decrease the reactivity of the
active intermediates. This inhibition is made worse by the crowding
around the active phosphorus reagent that occurs in the chemical
synthesis of RNA as a result of the protected 2'-hydroxyl (--OR in
the following scheme).
##STR00005##
While there are examples of phosphorus protecting groups in the
literature, there remains a need for novel phosphorus protecting
groups for polynucleotides, e.g. for use during polynucleotide
synthesis.
SUMMARY OF THE INVENTION
In certain embodiments of the invention, novel compositions having
a phosphorus group and a phosphorus protecting group bound to the
phosphorus group are provided, and methods of deprotecting the
phosphorus group are provided. In certain embodiments, the
phosphorus protecting group has the structure
##STR00006##
wherein: R is lower alkyl, modified lower alkyl, or alkyl; R.sup.i
and R.sup.ii are each independently selected from H, lower alkyl,
modified lower alkyl, alkyl, modified alkyl, or aryl; and
the broken line indicates a bond to the phosphorus group.
In typical embodiments, the phosphorus protecting group is
characterized as being labile under conditions which include an
.alpha.-effect nucleophile. Also, in typical embodiments, methods
are provided comprising contacting a polynucleotide having a
phosphorus protecting group with a solution comprising an
.alpha.-effect nucleophile to result in cleavage of the phosphorus
protecting group.
Additional objects, advantages, and novel features of this
invention shall be set forth in part in the descriptions and
examples that follow and in part will become apparent to those
skilled in the art upon examination of the following specifications
or may be learned by the practice of the invention. The objects and
advantages of the invention may be realized and attained by means
of the materials and methods particularly pointed out in the
appended claims.
DETAILED DESCRIPTION
Before the invention is described in detail, it is to be understood
that unless otherwise indicated this invention is not limited to
particular materials, reagents, reaction materials, manufacturing
processes, or the like, as such may vary. It is also to be
understood that the terminology used herein is for purposes of
describing particular embodiments only, and is not intended to be
limiting. It is also possible in the present invention that steps
may be executed in different sequence where this is logically
possible. However, the sequence described below is preferred.
It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "an insoluble support" includes a
plurality of insoluble supports. Similarly, reference to "a
substituent", as in a compound substituted with "a substituent",
includes the possibility of substitution with more than one
substituent, wherein the substituents may be the same or different.
In this specification and in the claims that follow, reference will
be made to a number of terms that shall be defined to have the
following meanings unless a contrary intention is apparent:
A "nucleotide" refers to a sub-unit of a nucleic acid (whether DNA
or RNA or analogue thereof) which includes a phosphate group, a
sugar group and a heterocyclic base, as well as analogs of such
sub-units. A "nucleoside" references a nucleic acid subunit
including a sugar group and a heterocyclic base. A "nucleoside
moiety" refers to a portion of a molecule having a sugar group and
a heterocyclic base (as in a nucleoside); the molecule of which the
nucleoside moiety is a portion may be, e.g. a polynucleotide,
oligonucleotide, or nucleoside phosphoramidite. A "nucleobase"
references the heterocyclic base of a nucleoside or nucleotide. An
"optionally protected nucleobase" references the heterocyclic base
of a nucleoside or nucleotide, wherein the heterocyclic base
optionally has bound thereto a protecting group, e.g. bound to an
imine nitrogen of the heterocyclic base or to an exocyclic amine
group of the heterocyclic base. A "nucleotide monomer" refers to a
molecule which is not incorporated in a larger oligo- or
poly-nucleotide chain and which corresponds to a single nucleotide
sub-unit; nucleotide monomers may also have activating or
protecting groups, if such groups are necessary for the intended
use of the nucleotide monomer. A "polynucleotide moiety" references
a moiety that has at least two nucleotide subunits. A
"polynucleotide intermediate" references a molecule occurring
between steps in chemical synthesis of a polynucleotide, where the
polynucleotide intermediate is subjected to further reactions to
get the intended final product, e.g. a phosphite intermediate which
is oxidized to a phosphate in a later step in the synthesis, or a
protected polynucleotide which is then deprotected.
As used herein, polynucleotides include single or multiple stranded
configurations, where one or more of the strands may or may not be
completely aligned with another. The terms "polynucleotide" and
"oligonucleotide" are generic to polydeoxynucleotides (containing
2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to
any other type of polynucleotide having nucleotide subunits that
are N-glycosides of a purine or pyrimidine base, and to other
polymers in which the conventional backbone has been replaced with
a non-naturally occurring or synthetic backbone or in which one or
more of the conventional bases has been replaced with a
non-naturally occurring or synthetic base. An "oligonucleotide"
generally refers to a nucleotide multimer of about 2 to 200
nucleotides in length, while a "polynucleotide" includes a
nucleotide multimer having at least two nucleotides and up to
several thousand (e.g. 5000, or 10,000) nucleotides in length. It
will be appreciated that, as used herein, the terms "nucleoside",
"nucleoside moiety" and "nucleotide" will include those moieties
which contain not only the naturally occurring purine and
pyrimidine bases, e.g., adenine (A), thymine (T), cytosine (C),
guanine (G), or uracil (U), but also modified purine and pyrimidine
bases and other heterocyclic bases which have been modified (these
moieties are sometimes referred to herein, collectively, as "purine
and pyrimidine bases and analogs thereof). Such modifications
include, e.g., methylated purines or pyrimidines, acylated purines
or pyrimidines, and the like, or the addition of a protecting group
such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl,
benzoyl, or the like. The purine or pyrimidine base may also be an
analog of the foregoing; suitable analogs will be known to those
skilled in the art and are described in the pertinent texts and
literature. Common analogs include, but are not limited to,
1-methyladenine, 2-methyladenine, N.sup.6-methyladenine,
N.sup.6-isopentyladenine, 2-methylthio-N.sup.6-isopentyladenine,
N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine,
3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,
4-acetylcytosine, 1-methylguanine, 2-methylguanine,
7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,
8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
5-ethyluracil, 5-propyluracil, 5-methoxyuracil,
5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,
5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,
2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,
uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,
pseudouracil, 1-methylpseudouracil, queosine, inosine,
1-methylinosine, hypoxanthine, xanthine, 2-aminopurine,
6-hydroxyaminopurine, 6-thiopurine and 2,6-diaminopurine.
"Upstream" as used herein refers to the 5' direction along a
polynucleotide, e.g. a DNA or RNA molecule, relative to a point of
reference, e.g. a particular nucleotide moiety. "Downstream" refers
to the 3' direction along the polynucleotide relative to a point of
reference. Hence, a downstream moiety is located in the 3'
direction along the polynucleotide. Similarly, an upstream moiety
is located at (or is bound to) a nucleotide moiety that is located
in the 5' direction along the polynucleotide. The point of
reference will often be implied from context or may be generally
disposed relative to the referenced element, e.g. an upstream
element is located in the 5' direction of any downstream element,
and a downstream element is located in the 3' direction of any
upstream element. "3'-" and "5'-" relate to the position on a sugar
group of a nucleoside moiety and may reference the noted group most
closely related to the position, e.g. a 3'-O is the oxygen bound to
the 3'-C of the sugar group, further e.g. a 3'-phosphorus reference
the phosphorus most closely bound to the 3'-C of the sugar group
(i.e. the 3'-phosphorus is the phosphorus of the phospho group
bound to the 3'-C of the sugar group). A 3'-terminal nucleotide
moiety of a polynucleotide moiety is the nucleotide moiety at the
most 3' end of the polynucleotide moiety; in typical embodiments,
an adjacent nucleotide moiety is bound to the polynucleotide moiety
having the 3'-terminal nucleotide moiety via the 3'-terminal
nucleotide moiety, e.g. via an intemucleotide bond. Similarly, a
5'-terminal nucleotide moiety of a polynucleotide moiety is the
nucleotide moiety at the most 5' end of the polynucleotide moiety;
in typical embodiments, an adjacent nucleotide moiety is bound to
the polynucleotide moiety having the 5'-terminal nucleotide moiety
via the 5'-terminal nucleotide moiety, e.g. via an intemucleotide
bond.
The term "alkyl" as used herein, unless otherwise specified, refers
to a saturated straight chain, branched or cyclic hydrocarbon group
of 1 to 24, typically 1-12, carbon atoms, such as methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl,
cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,
3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term
"lower alkyl" intends an alkyl group of one to six carbon atoms,
and includes, for example, methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl,
neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl,
2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term "cycloalkyl"
refers to cyclic alkyl groups such as cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
The term "modified alkyl" refers to an alkyl group having from one
to twenty-four carbon atoms, and further having additional groups,
such as one or more linkages selected from ether-, thio-, amino-,
phospho-, oxo-, ester-, and amido-, and/or being substituted with
one or more additional groups including lower alkyl, aryl, alkoxy,
thioalkyl, hydroxyl, amino, amido, sulfonyl, thio, mercapto, imino,
halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone,
sulfoxy, phosphoryl, silyl, silyloxy, and boronyl. The term
"modified lower alkyl" refers to a group having from one to eight
carbon atoms and further having additional groups, such as one or
more linkages selected from ether-, thio-, amino-, phospho-, keto-,
ester- and amido-, and/or being substituted with one or more groups
including lower alkyl; aryl, alkoxy, thioalkyl, hydroxyl, amino,
amido, sulfonyl, thio, mercapto, imino, halo, cyano, nitro,
nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl,
silyl, silyloxy, and boronyl. The term "alkoxy" as used herein
refers to a substituent --O--R wherein R is alkyl as defined above.
The term "lower alkoxy" refers to such a group wherein R is lower
alkyl. The term "thioalkyl" as used herein refers to a substituent
--S--R wherein R is alkyl as defined above.
The term "alkenyl" as used herein, unless otherwise specified,
refers to a branched, unbranched or cyclic (e.g. in the case of C5
and C6) hydrocarbon group of 2 to 24, typically 2 to 12, carbon
atoms containing at least one double bond, such as ethenyl, vinyl,
allyl, octenyl, decenyl, and the like. The term "lower alkenyl"
intends an alkenyl group of two to eight carbon atoms, and
specifically includes vinyl and allyl. The term "cycloalkenyl"
refers to cyclic alkenyl groups.
The term "alkynyl" as used herein, unless otherwise specified,
refers to a branched or unbranched hydrocarbon group of 2 to 24,
typically 2 to 12, carbon atoms containing at least one triple
bond, such as acetylenyl, ethynyl, n-propynyl, isopropynyl,
n-butynyl, isobutynyl, t-butynyl, octynyl, decynyl and the like.
The term "lower alkynyl" intends an alkynyl group of two to eight
carbon atoms, and includes, for example, acetylenyl and propynyl,
and the term "cycloalkynyl" refers to cyclic alkynyl groups.
The term "aryl" as used herein refers to an aromatic species
containing 1 to 5 aromatic rings, either fused or linked, and
either unsubstituted or substituted with 1 or more substituents
typically selected from the group consisting of lower alkyl,
modified lower alkyl, aryl, aralkyl, lower alkoxy, thioalkyl,
hydroxyl, thio, mercapto, amino, imino, halo, cyano, nitro,
nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl,
silyl, silyloxy, and boronyl; and lower alkyl substituted with one
or more groups selected from lower alkyl, alkoxy, thioalkyl,
hydroxyl thio, mercapto, amino, imino, halo, cyano, nitro, nitroso,
azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl,
silyloxy, and boronyl. Typical aryl groups contain 1 to 3 fused
aromatic rings, and more typical aryl groups contain 1 aromatic
ring or 2 fused aromatic rings. Aromatic groups herein may or may
not be heterocyclic. The term "aralkyl" intends a moiety containing
both alkyl and aryl species, typically containing less than about
24 carbon atoms, and more typically less than about 12 carbon atoms
in the alkyl segment of the moiety, and typically containing 1 to 5
aromatic rings. The term "aralkyl" will usually be used to refer to
aryl-substituted alkyl groups. The term "aralkylene" will be used
in a similar manner to refer to moieties containing both alkylene
and aryl species, typically containing less than about 24 carbon
atoms in the alkylene portion and 1 to 5 aromatic rings in the aryl
portion, and typically aryl-substituted alkylene. Exemplary aralkyl
groups have the structure --(CH2)j-Ar wherein j is an integer in
the range of 1 to 24, more typically 1 to 6, and Ar is a monocyclic
aryl moiety.
The term "heterocyclic" refers to a five- or six-membered
monocyclic structure or to an eight- to eleven-membered bicyclic
structure which is either saturated or unsaturated. The
heterocyclic groups herein may be aliphatic or aromatic. Each
heterocyclic group consists of carbon atoms and from one to four
heteroatoms selected from the group consisting of nitrogen, oxygen
and sulfur. As used herein, the term "nitrogen heteroatoms"
includes any oxidized form of nitrogen and the quaternized form of
nitrogen. The term "sulfur heteroatoms" includes any oxidized form
of sulfur. Examples of heterocyclic groups include purine,
pyrimidine, piperidinyl, morpholinyl and pyrrolidinyl.
"Heterocyclic base" refers to any natural or non-natural
heterocyclic moiety that can participate in base pairing or base
stacking interaction on an oligonucleotide strand.
An "internucleotide bond" refers to a chemical linkage between two
nucleoside moieties, such as a phosphodiester linkage in nucleic
acids found in nature, or such as linkages well known from the art
of synthesis of nucleic acids and nucleic acid analogues. An
intemucleotide bond may comprise a phosphate or phosphite group,
and may include linkages where one or more oxygen atoms of the
phosphate or phosphite group are either modified with a substituent
or replaced with another atom, e.g. a sulfur atom or the nitrogen
atom of a mono- or di-alkyl amino group.
The term "halo" or "halogen" is used in its conventional sense to
refer to a chloro, bromo, fluoro or iodo substituent.
When used herein, the terms "hemiacetal", "thiohemiacetal",
"acetal", and "thioacetal" are art-recognized, and refer to a
chemical moiety in which a single carbon atom is geminally
disubstituted with either two oxygen atoms or a combination of an
oxygen atom and a sulfur atom. In addition, when using the terms,
it is understood that the carbon atom may be geminally
disubstituted by two carbon atoms, forming ketal compounds. The
terms "hemiacetal", "thiohemiacetal", "acetal", and "thioacetal"
are generic to the corresponding ketal compounds (respectively,
"hemiketal", "thiohemiketal", "ketal", and "thioketal").
A "phospho" group includes a phosphodiester, phosphotriester, and
H-phosphonate groups. In the case of either a phospho or phosphite
group, a chemical moiety other than a substituted 5-membered furyl
ring may be attached to O of the phospho or phosphite group which
links between the furyl ring and the P atom.
By "protecting group" as used herein is meant a species which
prevents a portion of a molecule from undergoing a specific
chemical reaction, but which is removable from the molecule
following completion of that reaction, as taught for example in
Greene, et al., "Protective Groups in Organic Synthesis," John
Wiley and Sons, Second Edition, 1991. A "peroxyanion-labile linking
group" is a linking group that releases a linked group when
contacted with a solution containing peroxyanions. Similarly, a
"peroxyanion-labile protecting group" is a protecting group that is
removed from the corresponding protected group when contacted with
a solution containing peroxyanions. As used herein, "2'-O
protecting groups" or "2'-hydroxyl protecting groups" are
protecting groups which protect the 2'-hydroxyl groups of the
polynucleotide (e.g. bound to the 2'-O). As used herein,
"phosphorus protecting group" (sometimes referenced as "phosphate
protecting group") references a protecting group which protects a
phosphorus group (e.g. is bound to a phosphorus group wherein the
phosphorus group is attached to a sugar moiety of, e.g. a
nucleotide, a nucleoside phosphoramidite, a polynucleotide
intermediate, or a polynucleotide). As used herein, "cleaving",
"cleavage", "deprotecting", "releasing", or like terms when used in
reference to a protecting group refers to breaking a bond via which
the protecting group is bound to the protected group, resulting in
the cleaved protecting group and the deprotected moiety (the moiety
that was the protected group when bound to the protecting
group).
The term "electron withdrawing" denotes the tendency of a
substituent to attract valence electrons of the molecule of which
it is a part, i.e., an electron-withdrawing substituent is
electronegative with respect to neighboring atoms. A quantification
of the level of electron-withdrawing capability is given by the
Hammett sigma constant. This well known constant is described in
many references, for instance, March, Advanced Organic Chemistry
251-59, McGraw Hill Book Company, New York, (1977). Exemplary
electron-withdrawing groups include nitro, acyl, formyl, sulfonyl,
trifluoromethyl, cyano, chloride, and the like.
The term "electron-donating" refers to the tendency of a
substituent to repel valence electrons from neighboring atoms,
i.e., the substituent is less electronegative with respect to
neighboring atoms. Exemplary electron-donating groups include
amino, methoxy, alkyl (including alkyl having a linear or branched
structure, alkyl having one to eight carbons), cycloalkyl
(including cycloalkyl having four to nine carbons), and the
like.
The term "alpha effect," as in an "alpha effect nucleophile" in a
deprotection/oxidation agent, is used to refer to an enhancement of
nucleophilicity that is found when the atom adjacent a nucleophilic
site bears a lone pair of electrons. As the term is used herein, a
nucleophile is said to exhibit an "alpha effect" if it displays a
positive deviation from a Bronsted-type nucleophilicity plot. Hoz
et al. (1985) Israel J. Chem. 26:313. See also, Aubort et al.
(1970) Chem. Comm. 1378; Brown et al. (1979) J. Chem. Soc. Chem.
Comm.171; Buncel et al. (1982) J. Am. Chem. Soc. 104:4896; Edwards
et al. (1962) J. Am. Chem. Soc. 84:16; Evanseck et al. (1987) J.
Am. Chem Soc. 109:2349. The magnitude of the alpha effect is
dependent upon the electrophile which is paired with the specific
nucleophile. McIsaac, Jr. et al. (1972), J. Org. Chem. 37:1037.
Peroxy anions are example of nucleophiles which exhibit strong
alpha effects.
"Moiety" and "group" are used interchangeably herein to refer to a
portion of a molecule, typically having a particular functional or
structural feature, e.g. a linking group (a portion of a molecule
connecting two other portions of the molecule), or an ethyl moiety
(a portion of a molecule with a structure closely related to
ethane).
"Linkage" as used herein refers to a first moiety bonded to two
other moieties, wherein the two other moieties are linked via the
first moiety. Typical linkages include ether (--O--), oxo
(--C(O)--), amino (--NH--), amido (--N--C(O)--), thio (--S--),
phosphate (--PO.sub.4H--), ester (--O--C(O)--).
"Bound" may be used herein to indicate direct or indirect
attachment. In the context of chemical structures, "bound" (or
"bonded") may refer to the existence of a chemical bond directly
joining two moieties or indirectly joining two moieties (e.g. via a
linking group). The chemical bond may be a covalent bond, an ionic
bond, a coordination complex, hydrogen bonding, van der Waals
interactions, or hydrophobic stacking, or may exhibit
characteristics of multiple types of chemical bonds. In certain
instances, "bound" includes embodiments where the attachment is
direct and also embodiments where the attachment is indirect.
"Functionalized" references a process whereby a material is
modified to have a specific moiety bound to the material, e.g. a
molecule or substrate is modified to have the specific moiety; the
material (e.g. molecule or support) that has been so modified is
referred to as a functionalized material (e.g. functionalized
molecule or functionalized support).
The term "substituted" as used to describe chemical structures,
groups, or moieties, refers to the structure, group, or moiety
comprising one or more substituents. As used herein, in cases in
which a first group is "substituted with" a second group, the
second group is attached to the first group whereby a moiety of the
first group (typically a hydrogen) is replaced by the second
group.
"Substituent" references a group that replaces another group in a
chemical structure. Typical substituents include nonhydrogen atoms
(e.g. halogens), functional groups (such as, but not limited to
amino, sulfhydryl, carbonyl, hydroxyl, alkoxy, carboxyl, silyl,
silyloxy, phosphate and the like), hydrocarbyl groups, and
hydrocarbyl groups substituted with one or more heteroatoms.
Exemplary substituents include alkyl, lower alkyl, aryl, aralkyl,
lower alkoxy, thioalkyl, hydroxyl, thio, mercapto, amino, imino,
halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone,
sulfoxy, phosphoryl, silyl, silyloxy, boronyl, and modified lower
alkyl.
A "group" includes both substituted and unsubstituted forms.
Typical substituents include one or more lower alkyl, amino, imino,
amido, alkylamino, arylamino, alkoxy, aryloxy, thio, alkylthio,
arylthio, alkyl; aryl, thioalkyl, hydroxyl, mercapto, halo, cyano,
nitro, nitroso, azido, carboxy, sulfide, sulfonyl, sulfoxy,
phosphoryl, silyl, silyloxy, and boronyl optionally substituted on
one or more available carbon atoms with a nonhydrocarbyl
substituent such as cyano, nitro, halogen, hydroxyl, sulfonic acid,
sulfate, phosphonic acid, phosphate, or phosphonate or the like.
Any substituents are typically chosen so as not to substantially
adversely affect reaction yield (for example, not lower it by more
than 20% (or 10%, or 5% or 1%) of the yield otherwise obtained
without a particular substituent or substituent combination).
As used herein, "dissociation constant", e.g. an acid dissociation
constant, has its conventional definition as used in the chemical
arts and references a characteristic property of a molecule having
a tendency to lose a hydrogen ion. The value of a dissociation
constant mentioned herein is typically expressed as a negative
log.sub.10 value, i.e. a pKa (for an acid dissociation
constant).
Hyphens, or dashes, are used at various points throughout this
specification to indicate attachment, e.g. where two named groups
are immediately adjacent a dash in the text, this indicates the two
named groups are attached to each other. Similarly, a series of
named groups with dashes between each of the named groups in the
text indicates the named groups are attached to each other in the
order shown. Also, a single named group adjacent a dash in the text
indicates the named group is typically attached to some other,
unnamed group. In some embodiments, the attachment indicated by a
dash may be, e.g. a covalent bond between the adjacent named
groups. In some other embodiments, the dash may indicate indirect
attachment, i.e. with intervening groups between the named groups.
At various points throughout the specification a group may be set
forth in the text with or without an adjacent dash, (e.g. amido or
amido-, further e.g. Lnk, Lnk- or -Lnk-) where the context
indicates the group is intended to be (or has the potential to be)
bound to another group; in such cases, the identity of the group is
denoted by the group name (whether or not there is an adjacent dash
in the text). Note that where context indicates, a single group may
be attached to more than one other group (e.g. where a linkage is
intended, such as linking groups).
"Optional" or "optionally" means that the subsequently described
circumstance may or may not occur, so that the description includes
instances where the circumstance occurs and instances where it does
not. For example, the phrase "optionally substituted" means that a
non-hydrogen substituent may or may not be present, and, thus, the
description includes structures wherein a non-hydrogen substituent
is present and structures wherein a non-hydrogen substituent is not
present. At various points herein, a moiety may be described as
being present zero or more times: this is equivalent to the moiety
being optional and includes embodiments in which the moiety is
present and embodiments in which the moiety is not present. If the
optional moiety is not present (is present in the structure zero
times), adjacent groups described as linked by the optional moiety
are linked to each other directly. Similarly, a moiety may be
described as being either (1) a group linking two adjacent groups,
or (2) a bond linking the two adjacent groups: this is equivalent
to the moiety being optional and includes embodiments in which the
moiety is present and embodiments in which the moiety is not
present. If the optional moiety is not present (is present in the
structure zero times), adjacent groups described as linked by the
optional moiety are linked to each other directly.
We have now developed compositions which have phosphorus protecting
groups. The phosphorus protecting groups are characterized as being
labile upon contact with an .alpha.-effect nucleophile. Thus, in
certain embodiments, methods of cleaving the phosphorus protecting
groups using an .alpha.-effect nucleophile are provided. In various
embodiments of the invention, novel compositions comprising a
polynucleotide bound to a phosphorus protecting group are provided.
In certain embodiments, novel compositions comprising a nucleoside
phosphoramidite having a phosphorus protecting group are provided.
In some embodiments, methods of removing a phosphorus protecting
group are provided.
Accordingly, in certain embodiments of the present invention, a
composition is provided having a structure selected from structure
(I) or structure (II):
##STR00007## or a salt, conjugate base, or ionized form
thereof,
wherein: HeteroBase* is an optionally protected nucleobase; A is H,
OH, or a 2'-hydroxyl protecting group; R is lower alkyl, modified
lower alkyl, or alkyl; R.sup.i and R.sup.ii are each independently
selected from H, lower alkyl, modified lower alkyl, alkyl, modified
alkyl, or aryl; R.sup.iii and R.sup.iv are each independently
selected from lower alkyl, or R.sup.iii and R.sup.iv taken together
are cycloalkyl; and R.sup.v is H, a hydroxyl protecting group, a
nucleotide moiety, or an oligonucleotide moiety.
In embodiments in which R.sup.v is a hydroxyl protecting group, the
hydroxyl protecting group at the 3' or 5' position (R.sup.v) may be
any hydroxyl protecting group known in the art of polynucleotide
synthesis. The hydroxyl protecting group at the 3' or 5' position
should be selected to be compatible with the intended use of the
composition, e.g. compatible with a selected synthesis method.
Particularly contemplated are protecting groups known for their use
in the 4-step phosphoramidite synthesis methods, e.g. trityl or
modified trityl (e.g. monomethoxytrityl, dimethoxytrityl, etc.)
In typical embodiments, R.sup.v is a hydroxyl protecting group, and
the composition of structure (I) or structure (II) is a nucleoside
3'-phosphoramidite monomer or a nucleoside 5'-phosphoramidite
monomer, respectively.
In certain embodiments, R.sup.v is a nucleotide moiety or an
oligonucleotide moiety, and the composition of structure (I) or
structure (II) is an oligonucleotide phosphoramidite. Such
embodiments typically arise in certain polynucleotide synthesis
schemes which involve adding small oligonucleotides during each
round of synthesis (rather than a single nucleotide), or may be
used to attach an oligonucleotide to a substrate or to another
oligonucleotide.
In particular embodiments, a composition having a structure
selected from structure (I) or structure (II) is provided in which
A is a 2'-hydroxyl protecting group, R is selected from methyl,
ethyl, n-propyl, or isopropyl; R.sup.i and R.sup.ii are each
independently selected from H, methyl, ethyl, n-propyl, or
isopropyl; R.sup.iii and R.sup.iv are each isopropyl; and R.sup.v
is a hydroxyl protecting group. In certain embodiments, R.sup.i and
R.sup.ii are each independently selected from H, lower alkyl,
alkyl, or aryl.
In certain embodiments, a polynucleotide is provided having a
phosphorus protecting group in accordance with the present
invention. Such a polynucleotide typically has a series of
nucleotide subunits bound to each other, at least one of the
nucleotide subunits having the structure (III):
##STR00008##
wherein: HeteroBase* is an optionally protected nucleobase; A is H,
OH, or a 2'-hydroxyl protecting group; R is lower alkyl, modified
lower alkyl, or alkyl; R.sup.i and R.sup.ii are each independently
selected from H, lower alkyl, modified lower alkyl, alkyl, modified
alkyl, or aryl; and the broken lines indicate sites of attachment
to the remainder of the polynucleotide.
Regarding the nucleotide subunit having the structure (III), in
particular embodiments A is a 2'-hydroxyl protecting group, R is
selected from methyl, ethyl, n-propyl, or isopropyl; and R.sup.i
and R.sup.ii are each independently selected from H, methyl, ethyl,
n-propyl, or isopropyl. In certain embodiments, R.sup.i and
R.sup.ii are each independently selected from H, lower alkyl,
alkyl, or aryl.
In certain such embodiments, a polynucleotide is provided having
the structure (IV):
##STR00009##
wherein: HeteroBase* is an optionally protected nucleobase; A is H,
OH, or a 2'-hydroxyl protecting group; R is lower alkyl, modified
lower alkyl, or alkyl; R.sup.i and R.sup.ii are each independently
selected from H, lower alkyl, modified lower alkyl, alkyl, modified
alkyl, or aryl; PN3 is polynucleotide moiety; and PN5 is
polynucleotide moiety.
PN3 is an upstream polynucleotide moiety, i.e. PN3 is upstream of
the remainder of the polynucleotide having the structure (IV). The
upstream polynucleotide moiety PN3 has a 3'-terminal nucleotide
moiety, the 3'-terminal nucleotide moiety bound to the 5'-O of
structure (IV); i.e. the 3'-terminal nucleotide moiety has a
3'-phosphorus, the 5'-O of structure (IV) bound to the
3'-phosphorus of the 3'-terminal nucleotide moiety, i.e. to form a
phosphotriester linkage. PN5 is downstream polynucleotide moiety,
i.e PN5 is downstream of the remainder of the polynucleotide having
the structure (IV). The downstream polynucleotide moiety PN5 has a
5'-terminal nucleotide moiety, the 5'-terminal nucleotide moiety
bound to the indicated phosphate oxygen of structure (IV); i.e. the
5'-terminal nucleotide moiety has a 5'-carbon, the 5' carbon of the
5'-terminal nucleotide moiety bound to the indicated phosphate
oxygen of structure (IV), i.e. to form a phosphotriester linkage.
The upstream polynucleotide moiety PN3 may be any polynucleotide
moiety having at least two nucleotide subunits. Similarly, the
downstream polynucleotide moiety PN5 may be any polynucleotide
moiety having at least two nucleotide subunits. The upstream
polynucleotide moiety PN3 is attached to the downstream
polynucleotide moiety PN5 via a nucleotide subunit having the
structure (III).
The 2'-hydroxyl protecting group (designated "A" in structures (I),
(II), (III), and (IV) ) may be any hydroxyl protecting group known
in the art of polynucleotide synthesis. The 2'-hydroxyl protecting
group should be selected to be compatible with the intended use of
the composition, e.g. compatible with a selected synthesis method.
In this regard, "compatible" means that the protecting groups are
stable under conditions required, e.g. in the selected synthesis
method, labile under conditions required for deprotection, and
which do not otherwise significantly interfere with the intended
use. Particularly contemplated are the 2'-hydroxyl protecting
groups described in copending application filed on the same day as
the present application by Dellinger et al. entitled "Monomer
Compositions for the Synthesis of Polynucleotides, Methods of
Synthesis, and Methods of Deprotection" and, now U.S. patent
application Ser. No. 11/388,112, and U.S. Patent Application filed
by Dellinger et al. entitled "Solutions, Methods, and Processes for
Deprotection of Polynucleotides" and, now U.S. patent application
Ser. No. 11/387,369.
In certain embodiments, a polynucleotide is provided having a
plurality of nucleotide subunits, at least one of said plurality of
nucleotide subunits bound to a phosphorus protecting group, the
phosphorus protecting group having the structure (V):
##STR00010##
wherein: R is lower alkyl, modified lower alkyl, or alkyl; R.sup.i
and R.sup.ii are each independently selected from H, lower alkyl,
modified lower alkyl, alkyl, modified alkyl, or aryl; and the
broken line indicates a bond to said at least one of said plurality
of nucleotide subunits. The broken line in structure (V) indicates
the site at which the phosphorus protecting group is bound to a
phosphorus which is bound to a 5'-O, a 3'-O, or both a 5'-O and a
3'-O (e.g. the phosphorus part of an intemucleotide bond).
In particular embodiments that have a phosphorus protecting group
having the structure (V), R may be selected from methyl, ethyl,
n-propyl, or isopropyl; and R.sup.i and R.sup.ii may each be
independently selected from H, methyl, ethyl, n-propyl, or
isopropyl. In certain embodiments, R.sup.i and R.sup.ii are each
independently selected from H, lower alkyl, alkyl, or aryl.
In particular embodiments, more than 50% of all of the nucleotide
subunits of a polynucleotide are bound to a phosphorus protecting
group according to the present invention, and contacting the
polynucleotide with a solution comprising an .alpha.-effect
nucleophile as described herein results in cleavage of the
phosphorus protecting groups to result in the deprotected
polynucleotide (the polynucleotide from which phosphorus protecting
groups have been removed).
The polynucleotide may be any polynucleotide, for example DNA, RNA,
a polynucleotide analog, a modified polynucleotide, a
polynucleotide having protecting groups (e.g. protecting groups
bound to the amine groups of nucleobases, protecting groups bound
to the phosphate groups of the polynucleotide, protecting groups
which protect hydroxyl groups of the polynucleotide (e.g. bound to
the 2'-O, 3'-O, or 5'-O), or other protecting groups).
In certain embodiments the polynucleotide may be attached to the
substrate, e.g. via a cleavable linker. The polynucleotide may be
synthesized in situ (e.g. synthesized one nucleotide at a time
using polynucleotide synthesis schemes well known in the art) or
may be separately synthesized and then attached to the substrate.
The polynucleotide may generally be attached to the substrate via
any available site of the polynucleotide, e.g. at the 2'-O, the
3'-O, the 5'-O, an amino group of a nucleobase, or any other site.
Typically, the polynucleotide is attached to the substrate at the
2'-O or the 3'-O, less typically at the 5'-O or at an amino group
of a nucleobase.
In certain embodiments, the polynucleotide may be attached to the
substrate via a linking group, which may be any group that is bound
to both the substrate and the polynucleotide and which doesn't
interfere with the manufacture or use of the polynucleotide. A
typical linking group may be selected from (1) a lower alkyl group;
(2) a modified lower alkyl group in which one or more linkages
selected from ether-, thio-, amino-, oxo-, ester-, and amido- is
present; (3) a modified lower alkyl substituted with one or more
groups including lower alkyl; aryl, aralkyl, alkoxyl, thioalkyl,
hydroxyl, amino, amido, sulfonyl, halo; or (4) a modified lower
alkyl substituted with one or more groups including lower alkyl;
alkoxyl, thioalkyl, hydroxyl, amino, amido, sulfonyl, halo, and in
which one or more linkages selected from ether-, thio-, amino-,
oxo-, ester-, and amido- is present. The linking group --Lnk-- may
be bonded to the substrate at any position of the linking group
--Lnk-- available to bind to the substrate. Similarly, the linking
group --Lnk-- may be bonded to the adjacent polynucleotide at any
position of the linking group --Lnk-- available to bind to the
adjacent polynucleotide. In certain embodiments, the linking group
--Lnk-- is a single methylene group, --CH.sub.2--, or may be an
alkyl group or modified alkyl group up to about 24 carbons long
(and which may be straight-chain or branched-chain). In certain
such embodiments, one or more linkages selected from ether-, oxo-,
thio-, and amino- is present in the straight-or branched chain
modified alkyl group. In an embodiment, the linking group --Lnk--
comprises optionally substituted ethoxy, propoxy, or butoxy groups
(i.e. may include the structure --{(CH.sub.2)m--O}n--, wherein m is
a integer selected from 2, 3, 4, and n is a integer selected from
1,2,3, 4, 5, 6). In an embodiment, the linking group --Lnk-- has
the structure --(CH.sub.2)m-Lkg-(CH.sub.2)n--, wherein m and n are
integers independently selected from the range of 1 to about 12,
e.g. from the range of 2 to about 8, and Lkg is a linkage selected
from ether-, thio-, amino-, oxo-, ester-, and amido-.
In particular embodiments, the linking group --Lnk--has a first
terminal site and a second terminal site. In such embodiments, the
linking group --Lnk--is bound to the substrate at the first
terminal site, and the linking group --Lnk--is bound to the
cleavable linker at the second terminal site. The first and second
terminal sites will depend on the design of the linking group
taking into consideration, for example, the method used to attach
the cleavable linker to the substrate.
The substrate may have a variety of forms and compositions. The
substrate may derive from naturally occurring materials, naturally
occurring materials that have been synthetically modified, or
synthetic materials. Examples of suitable support materials
include, but are not limited to, nitrocellulose, glasses, silicas,
teflons, and metals (e.g., gold, platinum, and the like). Suitable
materials also include polymeric materials, including plastics (for
example, polytetrafluoroethylene, polypropylene, polystyrene,
polycarbonate, and blends thereof, and the like), polysaccharides
such as agarose (e.g., that available commercially as
Sepharose.RTM., from Pharmacia) and dextran (e.g., those available
commercially under the tradenames Sephadex.RTM. and Sephacyl.RTM.,
also from Pharmacia), polyacrylamides, polystyrenes, polyvinyl
alcohols, copolymers of hydroxyethyl methacrylate and methyl
methacrylate, and the like.
The polynucleotide may be bound directly to the substrate (e.g. to
the surface of the substrate, e.g. to a functional group on the
surface) or indirectly bound to the substrate, e.g. via one or more
intermediate moieties (e.g. linking groups) and/or surface
modification layer on the substrate. The nature of the site on the
substrate to which the polynucleotide is attached (e.g. directly or
via a linking group) is not essential to the present invention, as
any known coupling chemistry compatible with the substrate (i.e.
which doesn't result in significant degradation of the substrate)
may be used to couple to the cleavable linker. As such, various
strategies of coupling the polynucleotide to a substrate using
functional groups on the substrate are known in the art and may be
employed advantageously in the disclosed methods. Typical
strategies require a complementary reactive group on the
polynucleotide (or linking group bound to the polynucleotide) or
are selected based on moieties already present on the
polynucleotide (or linking group bound to the polynucleotide) (e.g.
amino groups, hydroxyl groups, or other functional groups), for
example an active group on the substrate that is capable of
reacting with a corresponding reactive group attached to the
polynucleotide (or linking group bound to the polynucleotide) to
result in the polynucleotide bound to the substrate.
Accordingly, in certain embodiments of the present invention, a
method is provided wherein the method includes: contacting a
polynucleotide with a solution comprising an .alpha.-effect
nucleophile, wherein the polynucleotide comprises a plurality of
nucleotide subunits, at least one of said plurality of nucleotide
subunits bound to a phosphorus protecting group, the phosphorus
protecting group having the structure (V):
##STR00011##
wherein: R is lower alkyl, modified lower alkyl, or alkyl; R.sup.i
and R.sup.ii are each independently selected from H, lower alkyl,
modified lower alkyl, alkyl, modified alkyl, or aryl; and the
broken line indicates a bond to said at least one of said plurality
of nucleotide subunits; said contacting resulting in cleavage of
the phosphorus protecting group from said at least one of said
plurality of nucleotide subunits.
Thus, in particular embodiments of the present invention, a method
of deprotecting a polynucleotide is provided, the method comprising
contacting the polynucleotide with a solution comprising an
.alpha.-effect nucleophile, wherein the polynucleotide comprises a
phosphorus group and a phosphorus protecting group bound thereto,
the phosphorus protecting group having the structure (V):
##STR00012##
wherein: R is lower alkyl, modified lower alkyl, or alkyl; R.sup.i
and R.sup.ii are each independently selected from H, lower alkyl,
modified lower alkyl, alkyl, modified alkyl, or aryl; and the
broken line indicates a bond to said phosphorus group; said
contacting resulting in cleavage of the phosphorus protecting group
from said phosphorus group.
As mentioned above, embodiments of the present disclosure include
methods for deprotecting a polynucleotide, wherein the
polynucleotide includes a phophorus protecting group such as those
described herein. In particular embodiments, the method includes
contacting the polynucleotide with a solution of an .alpha.-effect
nucleophile (e.g., a peroxyanion solution), wherein the
.alpha.-effect nucleophile has a pKa (negative log.sub.10 of the
acid dissociation constant) of about 4 to 13. In addition, the
solution is at a pH of about 6to 11.
In particular embodiments, a polynucleotide is contacted with a
solution of peroxyanions to result in deprotection of the
polynucleotide (e.g. cleavage of the phophorus protecting group
from the polynucleotide), wherein the peroxyanions have a pKa
within the range of about 4 to 12, at neutral to mildly basic pH
(e.g. the pH typically is in the range from about 6 to about
11).
In typical embodiments, the conditions employed for deprotection
include contacting the polynucleotide with the solution of the
.alpha.-effect nucleophile for time sufficient to result in
cleavage of the phophorus protecting group. Typical times
(duration) for the cleavage reaction range from about 15 minutes to
about 24 hour, although times outside this range may be used.
Typically the duration of the contacting is in the range from about
30 minutes to about 16 hours, e.g. from about 45 minutes to about
12 hours, from about 1 hour to about 8 hours, or from about 1 hour
to about 4 hours.
One advantage of using a neutral to mildly basic (e.g. pH in the
range from about 6 to about 11) solution including an
.alpha.-effect nucleophile is that the solution including an
.alpha.-effect nucleophile is compatible with standard
phosphoramidite methods for polynucleotide synthesis. Further, the
deprotected polynucleotides are stable and show little or no
degradation for an extended period of time when stored in the
solution including the .alpha.-effect nucleophile.
In general, the solution including the .alpha.-effect nucleophile
can be a predominantly buffered aqueous solution or buffered
aqueous/organic solution. In certain embodiments, it is convenient
and cost effective to recover the deprotected polynucleotide from
the mixture of deprotected polynucleotide, cleaved phosphorus
protecting groups, and solution of .alpha.-effect nucleophile by
simple precipitation of the desired polynucleotides directly from
the mixture by addition of ethanol to the mixture. Under these
conditions, the polynucleotide is pelleted to the bottom of a
centrifuge tube and the supernatant containing the .alpha.-effect
nucleophile removed by simply pouring off the supernatant and
rinsing the pellet with fresh ethanol. The deprotected
polynucleotide is then isolated by resuspending in a typical buffer
for chromatographic purification or direct usage in the biological
experiment of interest. Because of the nature of most
.alpha.-effect nucleophiles, removal from the desired deprotected
polynucleotide products is easy, quick, and effective using the
ethanol precipitation method. Any other methods of recovering the
deprotected polynucleotides may be employed, such as using Micro
Bio-Spin.TM. chromatography columns (BioRad, Hercules, Calif.) for
cleanup and purification of polynucleotides (used according to
product insert instructions).
The solution including the .alpha.-effect nucleophile typically may
have a pH in the range of about 4 to 11, about 5 to 11, about 6 to
11, about 7 to 11, about 8 to 11, about 4 to 10, about 5 to 10,
about 6 to 10, about 7 to 10, or about 8 to 10. In particular
embodiments the solution has a pH of about 7 to 10. It should also
be noted that the pH is dependent, at least in part, upon the
.alpha.-effect nucleophile in the solution and the protecting
groups on the polynucleotide. Appropriate adjustments to the pH can
be made to the solution to accommodate the .alpha.-effect
nucleophile.
The .alpha.-effect nucleophiles can include, but are not limited
to, peroxyanions, hydroxylamine derivatives, hydroximic acid and
derivatives thererof, hydroxamic acid and derivatives thereof,
carbazide and semicarbazides and derivatives thereof. The
.alpha.-effect nucleophiles can include compounds such as, but not
limited to, hydrogen peroxide, peracids, perboric acid salts,
alkylperoxides, hydrogen peroxide salts, hydroperoxides,
butylhydroperoxide, benzylhydroperoxide, phenylhydroperoxide,
cumene hydroperoxide, performic acid, peracetic acid, perbenzoic
acid and substituted perbenzoic acids such as chloroperbenzoic
acid, perbutyric acid, tertiary-butylperoxybenzoic acid,
decanediperoxoic acid, other similar compounds, and all
corresponding salts, and combinations thereof. Hydrogen peroxide,
salts of hydrogen peroxide and mixtures of hydrogen peroxide and
performic acid are especially useful. Hydrogen peroxide, whose pKa
is around 11, is particularly useful in solutions above pH 9.0.
Below pH 9.0 there is no significant concentration of peroxyanion
to work as an effective nucleophile. Below pH 9.0 it is especially
useful to use mixtures of hydrogen peroxide and peracids. These
peracids can be preformed and added to the solution or they can be
formed in situ by the reaction of hydrogen peroxide and the
carboxylic acid or carboxylic acid salt. An example is that an
equal molar mixture of hydrogen peroxide and sodium formate can be
used at pH conditions below 9.0 as an effective .alpha.-effect
nucleophile solution where hydrogen peroxide alone is not provide a
high concentration of .alpha.-effect nucleophiles. The utility of
peracids tends to be dependent upon the pKa of the acid and size of
molecule: the higher the pKa of the acid the more useful as a
peroxyanion solution, the larger the size of the molecule the less
useful. Typically the pKa of the peracid is lower than the pH of
the desired peroxyanion solution.
The .alpha.-effect nucleophiles typically used in these reactions
are typically strong oxidants, therefore one should limit the
concentration of the reagent in the solution in order to avoid
oxidative side products where undesired. The .alpha.-effect
nucleophiles are typically less than 30% weight/vol of the
solution, more typically between 0.1% and 10% weight/vol of the
solution and most typically 3% to 7% weight/vol of the solution.
The typical 3% solution of hydrogen peroxide is about 1 molar
hydrogen peroxide. A solution of between 1 molar and 2 molar
hydrogen peroxide is typically useful. A typical solution of
hydrogen peroxide and performic acid is an equal molar mixture of
hydrogen peroxide and performic acid, both in the range of 1 to 2
molar. An example of an in situ prepared solution of performic acid
is 2 molar hydrogen peroxide and 2 molar sodium formate buffered at
pH 8.5.
In typical embodiments, the .alpha.-effect nucleophile is
characterized as having a pKa in the range from about 4 to 13,
about 4 to 12, about 4 to 11, about 5 to 13, about 5 to 12, about 5
to 11, about 6 to 13, about 6 to 12, about 6 to 11, about 7 to 13,
about 7 to 12, or about 7 to 11.
It should also be noted that the dissociation constant (the pKa) is
a physical constant that is characteristic of the specific
.alpha.-effect nucleophile. Chemical substitution and solvent
conditions can be used to raise or lower the effective dissociation
constant and therefore specifically optimize the conditions under
which the deprotection of the polynucleotide is performed (to
result in release of the phosphorus protecting group from the
polynucleotide, and, optionally, deprotection of other groups
protected by peroxyanion-labile protecting groups). Appropriate
selection of the .alpha.-effect nucleophile should be made
considering the other conditions of the method and the protecting
groups of the polynucleotide. In addition, mixtures of carboxylic
acids and hydroperoxides can be used to form salts of peracids in
situ.
As an example a solution of hydrogen peroxide can be used with a
solution of formic acid at pH conditions below 9.0. At pH
conditions less than 9.0 hydrogen peroxide is not significantly
ionized due to its pKa of around 11. At pH 7.0 only about 0.01% of
the hydrogen peroxide is in the ionized form of the .alpha.-effect
nucleophile. However, the hydrogen peroxide can react in situ with
the formic acid to form performic acid in a stable equilibrium. At
pH 7.0 the performic acid is significantly in the ionized form and
is an active .alpha.-effect nucleophile. The advantage of such an
approach is that solutions of performic acid tend to degrade
rapidly and stabilizers need to be added. The equilibrium that is
formed between the hydrogen peroxide solutions and the formic acid
helps stabilize the performic acid such that it can be used to
completely cleave the polynucleotides from the substrates prior to
degrading. Performic acid is especially useful in a buffered
mixture of hydrogen peroxide at pH 8.5 because the pKa of performic
acid is approximately 7.1. Peracetic acid is useful at pH 8.5 but
less useful than performic acid because the pKa of peracetic acid
is approximately 8.2. At pH 8.5 peracetic acid is only about 50%
anionic whereas at pH 8.5 performic acid is more than 90%
anionic.
In general, the pKa for the hydroperoxides is about 8 to 13. The
pKa for hydrogen peroxide is quoted to be about 10 to 12 depending
upon the method of analysis and solvent conditions. The pKa for the
alkylperoxides is about 8 to 14. The pKa for the peracids is about
3 to 9. In some embodiments in which the peroxyanion is
hydroperoxide, the solution is at pH of about 9 to 11, e.g. at a pH
of about 9 to about 10. In certain embodiments in which the
peroxyanion is an alkylperoxide, the solution is at pH of about 8
to 11. In embodiments where the peroxyanion is a peracid, the
solution is at pH of about 6 to 9. In addition, the peracid
typically has a pKa of about 4 to 10.
In addition, the aqueous buffer solution usually includes a buffer,
such as, but not limited to, tris(hydroxymethyl)aminomethane,
aminomethylpropanol, citric acid, N,N'-Bis(2-hydroxyethyl)glycine,
2-[Bis(2-hydroxyethyl)amino]-2-(hydroxy-methyl)-1,3-propanediol,
2-(Cyclohexylamino)ethane-2-sulfonic acid,
N-2-Hydroxyethyl)piperazine-N'-2-ethane sulfonic acid,
N-(2-Hydroxyethyl)piperazine-N'-3-propane sulfonic acid,
Morpholinoethane sulfonic acid, Morpholinopropane sulfonic acid,
Piperazine-N,N'-bis(2-ethane sulfonic acid),
N-Tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid,
N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid,
N-Tris(hydroxymethyl) methylglycine, and combinations thereof.
One significant potential advantage for removing the phosphorus
protecting group from the polynucleotide according to the present
methods is that the .alpha.-effect nucleophile solution can be
exploited to remove a variety of peroxyanion-labile protecting
groups at the same time and under the same conditions that are used
to cleave the phosphorus protecting group from the polynucleotide.
Thus, cleavage of the phosphorus protecting group from the
polynucleotide and deprotection of groups protected with
peroxyanion-labile protecting groups may be reduced to a single
step in which the phosphorus group deprotection and deprotection of
other sites on the polynucleotide (e.g. 2'-OH, nucleobase) occur at
essentially the same time in the same reaction mixture. These
advantages become even more significant if they are used with the
protecting groups described in the applications cited herein to
Dellinger et al. that were filed on the same day as the present
application; such protecting groups specifically provide for rapid
deprotection under the oxidative, nucleophilic conditions at
neutral to mildly basic pH.
Particularly contemplated is the use of the cleavable linkers
described in the two U.S. patent applications filed by Dellinger et
al. on the same day as the instant application (one entitled
"Cleavable Linkers for Polynucleotides" and, now U.S. patent
application Ser. No. 11/389,388; the second entitled "Thiocarbonate
Linkers for Polynucleotides" and now U.S. patent application Ser.
No. 11/751,692 in conjunction with phosphorus protecting groups
attached to the polynucleotide as described herein. Optionally,
peroxyanion-labile protecting groups may be attached, e.g. at the
2'-position of the nucleoside sugar of the individual nucleotide
subunits, at the exocyclic amine groups of the heterocyclic bases
of the polynucleotide, and/or at the imine groups of the
heterocyclic bases of the polynucleotide. In certain such
embodiments, contacting the polynucleotide with solution including
an .alpha.-effect nucleophile results in concurrent cleavage of the
polynucleotide from the substrate and deprotection of the
polynucleotide at the phosphate groups, as well as (optionally)
deprotection at the 2'-position of the nucleoside sugar, at the
exocyclic amine groups, and/or at the imine groups of the
heterocyclic bases.
In particular embodiments a polynucleotide having a phosphorus
protecting group as described herein has peroxyanion-labile
protecting groups on, e.g. the exocyclic amine groups of the
nucleobases. In some such embodiments, contacting the
polynucleotide with solution including an .alpha.-effect
nucleophile results in concurrent deprotection of the phosphate
groups and of the exocyclic amine groups. As another example, in
particular embodiments a polynucleotide having a phosphorus
protecting group as described herein has peroxyanion-labile
protecting groups on, e.g. the 2' position of the nucleoside sugar.
In certain such embodiments, contacting the polynucleotide with a
solution including an .alpha.-effect nucleophile results in
concurrent deprotection of the phosphate groups and of the 2'
position of the nucleoside sugar (e.g. resulting in a deprotected
2'-hydroxyl group). In a further example, a polynucleotide having a
phosphorus protecting group has peroxyanion-labile protecting
groups on, e.g. the 2' position of the nucleoside sugar and the
exocyclic amine groups. In certain such embodiments, contacting the
polynucleotide with a solution including an .alpha.-effect
nucleophile results in concurrent deprotection of the phosphate
groups, of the 2' position of the nucleoside sugar, and of the
exocyclic amine groups. In certain embodiments such as those
described above, the polynucleotide having a phosphorus protecting
group as described herein is bound to a substrate via a cleavable
linker, wherein the cleavable linker is labile upon contact with an
.alpha.-effect nucleophile. In certain such embodiments, contacting
the polynucleotide with a solution including an .alpha.-effect
nucleophile results in concurrent cleavage of the polynucleotide
from the substrate as well as deprotection of the phosphate groups,
and (optionally) of the 2' position of the nucleoside sugar, and
(optionally) of the exocyclic amine groups.
Structure (VII) serves to illustrate a portion of a polynucleotide
bound to a substrate, and illustrates that there are several sites
of the polynucleotide which may have protecting groups bound
thereto, including phosphorus protecting groups (designated R in
structure (VII), and sometimes referenced herein as "phosphate
protecting groups"), nucleobase protecting groups (designated R' in
structure (VII)); and 2'-hydroxyl protecting groups (designated R'
in structure (VII), and sometimes referenced herein as 2'-O
protecting groups). Note that structure (VII) only depicts two
nucleotide subunits, but that typically there will be many more
nucleotide subunits in the polynucleotide having the same general
structure as the nucleotide subunits depicted in structure (VII).
In structure (VII), B represents a nucleobase. It is contemplated
that, in particular embodiments, the phosphorus protecting groups
(R in structure (VII)) described herein may be labile under the
same conditions that result in removal of other protecting groups
(i.e. R' and/or R'') as well as cleavage of the cleavable linkers
(CLG in structure (VII) ) via which the polynucleotide is attached
to a substrate.
##STR00013##
In particular embodiments the polynucleotide has a plurality of
phosphate groups wherein each phosphate group is bound to a
phosphorus protecting group as described herein. The phosphorus
protecting group is labile upon being contacted with a solution of
an .alpha.-effect nucleophile (e.g. the phosphorus protecting group
is peroxyanion-labile). In certain embodiments, a cleavable linker
is used that is also labile upon being contacted with the solution
of the .alpha.-effect nucleophile. Thus, the cleavable linker may
be cleaved and the phosphate groups may undergo deprotection
concurrently upon being contacted with a solution comprising an
.alpha.-effect nucleophile. Examples of such cleavable linkers are
described in a copending application filed on the same day as the
instant application by Dellinger et al. entitled entitled
"Cleavable Linkers for Polynucleotides" and, now U.S. patent
application Ser. No. 11/389,388, and also in a copending
application filed on the same day as the instant application by
Dellinger et al. entitled "Thiocarbonate Linkers for
Polynucleotides" and, now U.S. patent application Ser. No.
11/751,692.
Thus, in particular embodiments, the present invention provides for
a method that includes: contacting a polynucleotide bound to a
substrate via a cleavable linker with a solution comprising an
.alpha.-effect nucleophile; wherein said cleavable linker is
characterized as being labile upon exposure to the .alpha.-effect
nucleophile; wherein the polynucleotide has a plurality of
phosphate groups; wherein each phosphate group of the plurality of
phosphate groups has a phosphorus protecting group bound thereto,
said phosphorus protecting group having the structure (V):
##STR00014##
wherein: R is lower alkyl, modified lower alkyl, or alkyl; R.sup.i
and R.sup.ii are each independently selected from H, lower alkyl,
modified lower alkyl, alkyl, modified alkyl, or aryl; and the
broken line indicates a bond to said phosphorus group;
said contacting resulting in concurrent cleavage of the
polynucleotide from the substrate and deprotection of each
phosphate group of the plurality of phosphate groups.
In particular embodiments the polynucleotide has a plurality of
nucleobases, wherein each nucleobase is bound to a nucleobase
protecting group. In certain such embodiments, the nucleobase
protecting group is labile under the same conditions as the
phosphorus protecting group (e.g. the nucleobase protecting group
is peroxyanion-labile). Thus, the nucleobases and the phosphate
groups may undergo deprotection concurrently upon being contacted
with a solution comprising an .alpha.-effect nucleophile. Any
nucleobase protecting group known in the art of polynucleotide
synthesis that is labile under conditions of being contacted with
the .alpha.-effect nucleophile may be used. Examples of such
nucleobase protecting groups are described in a copending
application filed on the same day as the instant application by
Dellinger et al. entitled "Monomer Compositions for the Synthesis
of Polynucleotides, Methods of Synthesis, and Methods of
Deprotection" and, now U.S. patent application Ser. No.
11/387,388.
Thus, in particular embodiments, the present invention provides for
a method that includes: contacting a polynucleotide with a solution
comprising an .alpha.-effect nucleophile; wherein the
polynucleotide has a plurality of nucleobases; wherein each
nucleobase of the plurality of nucleobases has a nucleobase
protecting group bound thereto, said nucleobase protecting group
characterized as being labile upon exposure to the .alpha.-effect
nucleophile; wherein the polynucleotide has a plurality of
phosphate groups; wherein each phosphate group of the plurality of
phosphate groups has a phosphorus protecting group bound thereto,
said phosphorus protecting group having the structure (V):
##STR00015##
wherein: R is lower alkyl, modified lower alkyl, or alkyl; R.sup.i
and R.sup.ii are each independently selected from H, lower alkyl,
modified lower alkyl, alkyl, modified alkyl, or aryl; and
the broken line indicates a bond to said phosphorus group;
said contacting resulting in concurrent deprotection of the
plurality of nucleobase and the plurality of phosphate groups.
In particular embodiments the polynucleotide has a plurality of
2'-O groups wherein each 2'-O group is bound to a 2'-O protecting
group (i.e. a 2'-hydroxyl protecting group). In certain such
embodiments, the 2'-O protecting group is labile under the same
conditions as the phosphorus protecting group (e.g. the 2'-O
protecting group is peroxyanion-labile). Thus, the phosphate groups
and the 2'-O groups may undergo deprotection concurrently upon
being contacted with a solution comprising an .alpha.-effect
nucleophile. Any 2'-O protecting group known in the art of
polynucleotide synthesis that is labile under conditions of being
contacted with the .alpha.-effect nucleophile may be used. Examples
of such 2'-O protecting groups are described in a copending
application filed on the same day as the instant application by
Dellinger et al. entitled "Monomer Compositions for the Synthesis
of Polynucleotides, Methods of Synthesis, and Methods of
Deprotection" and, now U.S. patent application Ser. No.
11/388,112.
Thus, in particular embodiments, the present invention provides for
a method that includes: contacting a polynucleotide with a solution
comprising an .alpha.-effect nucleophile; wherein the
polynucleotide has a plurality of 2'-O groups; wherein each 2'-O
group of the plurality of 2'-O groups has a 2'-O protecting group
bound thereto, said 2'-O protecting group characterized as being
labile upon exposure to the .alpha.-effect nucleophile; wherein the
polynucleotide has a plurality of phosphate groups; wherein each
phosphate group of the plurality of phosphate groups has a
phosphorus protecting group bound thereto, said phosphorus
protecting group having the structure (V):
##STR00016##
wherein: R is lower alkyl, modified lower alkyl, or alkyl; R.sup.i
and R.sup.ii are each independently selected from H, lower alkyl,
modified lower alkyl, alkyl, modified alkyl, or aryl; and
the broken line indicates a bond to said phosphorus group;
said contacting resulting in concurrent deprotection of the
plurality of 2'-O groups and the plurality of phosphate groups.
Furthermore, in certain embodiments, the polynucleotide includes a
plurality of phosphate groups, a plurality of nucleobases, and a
plurality of 2'-O groups. In some embodiments, the polynucleotide
also includes one or more (e.g. two or more, e.g. all three) types
of protecting groups selected from phosphorus protecting groups,
nucleobase protecting groups, or 2'-O protecting groups. Each
protecting group is bound to a corresponding site of the
polynucleotide (i.e. phosphorus protecting groups are bound to
phosphate groups, nucleobase protecting groups are bound to
nucleobases, and 2'-O protecting groups are bound to 2'-O groups).
In certain embodiments, the polynucleotide is bound to a substrate
via a cleavable linker, and the method provides for deprotection of
the polynucleotide and concurrent cleavage of the polynucleotide
from the substrate. The concurrent deprotection and cleavage may
include deprotection of: 1) the phosphate groups 2) the phosphate
groups and the nucleobases, 3) the phosphate groups and the 2'-O
groups; or 4) the phosphate groups, the nucleobases, and the 2'-O
groups.
Further Examples:
In some embodiments, the present invention provides compositions
having phosphorus protecting groups that are phosphate esters of
thiohemiacetals. Exemplary compositions are represented by the
general formula:
##STR00017##
wherein: R is lower alkyl, modified lower alkyl, or alkyl; R.sup.i
and R.sup.ii are each independently selected from H, lower alkyl,
modified lower alkyl, alkyl, modified alkyl, or aryl; R.sup.iii and
R.sup.iv are each independently selected from lower alkyl, or
R.sup.iii and R.sup.iv taken together are cycloalkyl; and Nu is a
nucleoside moiety.
Because of the electron donating nature of the thioacetal there is
an observed increased reactivity in coupling in the presence of
azole acid catalysts, as compared to cyanoethyl protected
phosphoramidites.
##STR00018##
This increased reactivity makes these reagents especially useful
for the synthesis of RNA as well as other oligonucleotides.
These novel phosphite reagents can either be prepared as
halophosphoramidites or bis-aminophosphodiamidites.
##STR00019##
These active phosphites can be reacted with protected nucleosides
to form novel phosphoramidite monomers. Halophosphoramidites are
reacted under anhydrous conditions with non-nucleophilic bases to
produce the desired protected nucleoside phosphoramidite
monomers.
##STR00020## (wherein HeteroBase is a nucleobase; A is a
2'-hydroxyl protecting group; APG is an optional nucleobase
protecting group; and R.sup.v is a hydroxyl protecting group).
The bis-aminophosphodiamidites are reacted using azole catalysts or
amine salts of azole catalysts (Barone A D, Tang J Y, Caruthers M
H, Nucleic Acids Res., 1984, 12(10), 4051-61) to produce the
desired protected nucleoside phosphoramidite monomers.
##STR00021##
A very useful species in this class is where R.sup.i and R.sup.ii
are both hydrogen. In this case the thiohemiacetal is quite stabile
and can be conveniently prepared from commercial reagents.
##STR00022##
The alcohol can then be converted into a reactive phosphate reagent
using standard methods
##STR00023##
In the case where R.sup.i and R.sup.ii are both hydrogen there is
less crowding around the active phosphorus center resulting in
higher coupling yields. In the case where R.sup.i and R.sup.ii are
both alkyl or aryl, the thiohemiacetal is less stabile and is best
made by condensation of a thiosilane on a ketone or aldehyde as
described by Evans et. al. (1997) J. Am. Chem. Soc.
99(15):5009-17:
##STR00024##
The trimethylsilyl hemithioacetal reagent can be reacted directly
with a chlorophosphine to yield the desired
bis-aminophosphodiamidites.
##STR00025##
Conversion to protected nucleoside phosphoramidite monomers as
described above provides reagents which can then be integrated into
the standard 4-step synthesis cycle:
##STR00026##
Post synthesis, the oligonucleotides are exposed to a solution of
peroxyanions at mildly basic pH. Without limiting the claimed
invention, it is believed that the peroxide first oxidizes the
sulfur to a sulfoxide then a sulfone. A peroxyanion then cleaves
the protecting group and liberates the phosphodiester
intemucleotide bond. Thus, contacting with the .alpha.-effect
nucleophile results in deprotection of the phosphate group, giving
the deprotected phosphate diester intemucleotide linkage.
##STR00027##
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of synthetic organic chemistry,
biochemistry, molecular biology, and the like, which are within the
skill of the art. Such techniques are explained fully in the
literature.
Experimental:
Synthesis of bis(N,N-diisopropylamino)chlorophosphine:
A 5 L three neck round bottom flask was equipped with a Friedrich's
condenser, a ground glass stirrer bearing, a silicon rubber septum,
and placed under dry argon. Two liters of anhydrous
diisopropylamine (1.6 kg, 15.9 mol) were added to the flask and
diluted by addition of two liters anhydrous acetonitrile. The
solution was mixed with a mechanical stirrer attached to a glass
rod and a Teflon blade. An ice-water bath was placed under the
flask and the solution allowed to cool with stirring for 30 min.
Phosphorus trichloride (313 g, 2.3 mol) was placed in a dry two
liter flask and one liter of anhydrous acetonitrile added. This
phosphorus trichloride solution was then added slowly by
cannulation to the vigorously stirred solution of diisopropylamine.
Once addition was complete, the ice bath was removed and the
reaction mixture stirred overnight. Complete conversion of the
phosphorus trichloride (.delta. 201 ppm) to product (.delta. 134
ppm) was monitored by .sup.31P NMR. The reaction mixture was
filtered to remove diisopropylamine hydrochloride and the
precipitate washed with anhydrous ether. The combined filtrates
were concentrated in vacuo to a semi-crystalline solid. Anhydrous
hexanes (1.5 L) were added to the flask and the mixture heated. The
hot liquid was filtered through a Schlenk filter funnel to remove
residual amine hydrochloride and the resulting clear liquid
concentrated in vacuo to half the original volume. The product was
then isolated by crystallization, filtration, and drying in vacuo
to yield 447 g (74% yield). .sup.31P NMR (CD.sub.3CN) .delta. 134.4
(s); Electron Impact Mass Spectrometry gave a molecular ion of 267
m/e.
Synthesis of bis(N,
N-diisopropylamino)methylthiomethylphosphite:
Acetic acid methylthiomethyl ester (50 mmol) was purchased from TCI
America (Portland, Oreg.) and dissolved in 200 mL of ether and 100
mL of a 1.0 M solution of KOH in water added. The reaction was
allowed to stir overnight and the ether solution separated, dried
over anhydrous sodium sulfate and evaporated to an oil to produce
the hemithioacetal. bis(N,N-Diisopropylamino)chlorophosphine (50
mmol) is dissolved in anhydrous acetonitrile with freshly
distilled, anhydrous, diisopropylethyl amine (60 mmol). The
hemithioacetal is added drop wise and the reaction stirred
overnight. The product is evaporated to an oil and the
phosphorodiamidite isolated by trituration into pentanes.
General Procedure for the Synthesis of Nucleoside
Phosphoramidites:
Protected deoxynucleosides were dissolved in anhydrous
dichloromethane at 0.05 to 0.1 M, depending upon their solubility,
and 1.2 molar equivalents of the appropriate phosphorodiamidite
added with stirring. Upon complete dissolution of the reaction
components, 0.8 molar equivalent tetrazole was added to the
reaction mixture as a 0.45 M solution in anhydrous acetonitrile.
The reaction mixture was allowed to stir for 24 h at room
temperature and then analyzed for extent of reaction by .sup.31P
NMR and silica gel TLC (eluted with ethyl acetate). The reaction
was determined to be complete by spot to spot conversion to a
faster eluting product on TLC and by complete loss of the
phosphorodiamdite 31P NMR signal. Upon completion, the reaction was
quenched by addition of 0.8 molar equivalents of anhydrous
triethylamine. After 5 min, the reaction mixture was concentrated
to a viscous oil in vacuo using a rotary evaporator. The viscous
oil was redissolved in a minimum volume of ethyl acetate and was
added to the top of a silica gel column pre-equilibrated with
various mixtures of ethyl acetate:hexanes. Isocratic elution of the
column with appropriate solvents for each preparation (monitored by
TLC) was used to collect the product. Fractions containing the
product were combined and concentrated to a foam in vacuo on a
rotary evaporator, redissolved in a minimal amount of anhydrous
dichloromethane, and added dropwise to rapidly stirring anhydrous
hexanes. The solid precipitate was isolated by filtration and dried
overnight in vacuo. The resulting white solids were analyzed by
.sup.31P NMR and FAB mass spectroscopy.
The instant specification is put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how to perform the methods and use the compositions
disclosed and claimed herein. Efforts have been made to ensure
accuracy with respect to numbers (e.g., amounts, temperature, etc.)
but some errors and deviations should be accounted for. Unless
indicated otherwise, parts are parts by weight, percents are
wt./wt., temperature is in .degree. C. and pressure is at or near
atmospheric. Standard temperature and pressure are defined as
20.degree. C. and 1 atmosphere.
A synthesis of reagents used in certain embodiments of the present
invention is now described. It will be readily apparent that the
reactions described herein may be altered, e.g. by using modified
starting materials to provide correspondingly modified products,
and that such alteration is within ordinary skill in the art. Given
the disclosure herein, one of ordinary skill will be able to
practice variations that are encompassed by the description herein
without undue experimentation.
While the foregoing embodiments of the invention have been set
forth in considerable detail for the purpose of making a complete
disclosure of the invention, it will be apparent to those of skill
in the art that numerous changes may be made in such details
without departing from the spirit and the principles of the
invention. Accordingly, the invention should be limited only by the
following claims.
All patents, patent applications, and publications mentioned herein
are hereby incorporated by reference in their entireties.
* * * * *